Biology Protocols

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Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)

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The support matrix for SDS-PAGE is polyacrylamide, as opposed to agarose, and is more thermostable, which is advantageous for this application where high voltage gradients are used to separate the constituent proteins in each sample

Proteins have a relative charge based upon their isoelectric point and molecular weight, which are determined by their structure. Therefore proteins of the same molecular weight, but different structures will migrate at different rates.

The sample to be analysed is mixed with sodium dodecyl sulphate (SDS) an anionic detergent. This denatures secondary and non–disulfide–linked tertiary structures, and applies a negative charge to each protein in proportion to its mass, by "wrapping around" the polypeptide backbone. This means that under these denaturing conditions, proteins can be assumed to migrate based on their size alone. To aid this unfolding process, the samples are also boiled briefly before loading onto the gel.

Alongside the sample fractions a protein ladder marker is also run to allow the size of the proteins in the experimental samples to be estimated.

 

Visualisation of SDS-PAGE is conducted using a solution containing coomassie brilliant blue, which binds proteins non-specifically. The staining solution also contains acetic acid and methanol, which fix the proteins for staining.

 


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